All cells store fatty acids in neutral lipids (triacylglycerols[TAG] & cholesterol esters) in discrete intracellular lipid storage droplets. In the general cell population, very small droplets transiently sequester fatty acids which are used for membrane biosynthesis and as a source of energy. In adipocytes, which store the main bodily energy reserves, fatty acids are mobilized from very large, TAG-rich, droplets and exported to other tissues. In steroidogenic cells, the droplets contain primarily cholesteryl esters, precursors for steroid hormone synthesis. Our research focuses on the surface composition of droplets and the mechanisms by which lipids are both deposited and hydrolyzed. We find that droplet surfaces in animal cells are coated by related proteins, the perilipins and adipocyte differentiation-related protein (ADRP). Despite its name, ADRP is expressed ubiquitously and occurs on the surface of droplets in nearly all cells examined. However, the three perilipin isoforms are expressed primarily in adipocytes and steroidogenic cells. These cell types are unique in that they use a cAMP/protein kinase A (PKA)-mediated process to hydrolyze their stored lipids. Since perilipins are polyphosphorylated by PKA in concert with the lipolytic reaction, we hypothesize that these proteins participate actively in lipid breakdown. Our data indicate that two PKA-mediated processes combine to promote lipid hydrolysis in adipocytes. First, hormone-sensitive lipase (HSL), the rate-limiting enzyme of lipolysis, rapidly translocates from the cytosol to the lipid droplet when cellular PKA activity is elevated. To determine if phosphorylation of HSL is required for the translocation reaction, we have assessed the consequences of mutation of the three known PKA sites in HSL: serines 563, 650 and 660. Mutation of 563 has no effect, but mutation of either 659 or 660 retards translocation, and mutation of both of these site abolishes the translocation reaction. Thus, phosphorylation of HSL is required for movement of the enzyme from the cytosol to the surface of the lipid storage droplet in adipose cells. The second phenomenon set in motion by PKA is phosphorylation of the perilipin that coats the lipid droplet. To study perilipin function, we have introduced perilipin A into cells, such as CHO fibroblasts, that normally do not express this protein. Ectopic perilipin A localizes exclusively to lipid droplets and increases TAG storage without increasing the rate of lipogenesis. Rather, perilipin A retards the rate of TAG hydrolysis. This protective, lipid-sparing effect is reversed when PKA activity is elevated. Moreover, mutational analysis of the PKA phosphorylation sites in perilipin reveals that perilipin phosphorylation is required to obtain the PKA-dependent increase in TAG hydrolysis in the fibroblastic cells. We conclude that in adipocytes, TAG hydrolysis is regulated by phosphorylation of both HSL and perilipin. Given the critical role of adipose neutral lipid reserves in times of need, it is not surprising that adipose cells evolved this sophisticated dual control system for parsimoniously meting out its supply of fatty acids. - adipose, lipolysis, perilipins, ADRP, hormone-sensitive lipase, lipid storage droplets